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Dietary supplementation with phytosterol and ascorbic acid reduces body mass accumulation and alters… Thornton, Sheila J; Wong, Ian T; Neumann, Rachel; Kozlowski, Petri; Wasan, Kishor M Jun 28, 2011

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RESEARCH Open AccessDietary supplementation with phytosterol andascorbic acid reduces body mass accumulationand alters food transit time in a diet-inducedobesity mouse modelSheila J Thornton*, Ian TY Wong, Rachel Neumann, Petri Kozlowski and Kishor M WasanAbstractPrevious research indicates that animals fed a high fat (HF) diet supplemented with disodium ascorbyl phytostanylphosphate (DAPP) exhibit reduced mass accumulation when compared to HF control. This compound is a water-soluble phytostanol ester and consists of a hydrophobic plant stanol covalently bonded to ascorbic acid (VitaminC). To provide insight into the mechanism of this response, we examined the in vivo effects of a high fat dietsupplemented with ascorbic acid (AA) in the presence and absence of unesterified phytosterols (PS), and set out toestablish whether the supplements have a synergistic effect in a diet-induced obesity mouse model. Our dataindicate that HF diet supplementation with a combination of 1% w/w phytosterol and 1% w/w ascorbic acidresults in reduced mass accumulation, with mean differences in absolute mass between PSAA and HF control of10.05%; and differences in mass accumulation of 21.6% (i.e. the PSAA group gained on average 21% less masseach week from weeks 7-12 than the HF control group). In our previous study, the absolute mass differencebetween the 2% DAPP and HF control was 41%, while the mean difference in mass accumulation between thetwo groups for weeks 7-12 was 67.9%. Mass loss was not observed in animals supplemented with PS or AA alone.These data suggest that the supplements are synergistic with respect to mass accumulation, and the esterificationof the compounds further potentiates the response. Our data also indicate that chronic administration of PS, bothin the presence and absence of AA, results in changes to fecal output and food transit time, providing insight intothe possibility of long-term changes in intestinal function related to PS supplementation.Keywords: Obesity, phytosterols, phytostanols, ascorbic acidIntroductionObesity is characterized by an over-accumulation of adi-pose tissue that is normally associated with excess calo-ric intake. Over the last few decades, research in thefield of obesity has lead us far from the concept of adi-pose tissue as an energy storage depot. We now under-stand that fat is a secretory tissue, producing a varietyof bioactive substances collectively referred to as adipo-cytokines. A number of these substances, notably tumornecrosis factor (TNF) alpha and plasminogen activatorinhibitor type 1, suggest that obesity is essentially aninflammatory disease, where excess adipose tissueinduces macrophage production and activates theimmune system without a legitimate pathogen [1].Numerous studies indicate that the dysregulation ofthese adipocytokines may directly contribute to obesity-related diseases [2]. In addition to adipocytokines, theproduction of reactive oxygen species (ROS) has alsobeen shown to be elevated in obesity and diabetes [3,4].Hyperglycemia, hyperlipidemia and hypercholesterole-mia, key clinical manifestations of obesity and diabetes,all promote ROS production through various pathways[5].Plant sterols (phytosterols) and their saturated deriva-tives, phytostanols, are among a growing list of dietarycomponents that exert a positive effect on hypercholes-terolemia. It is well established that certain plant sterols* Correspondence: thornton@zoology.ubc.caFaculty of Pharmaceutical Sciences, University of British Columbia, 2146 EastMall, Vancouver, BC, CanadaThornton et al. Lipids in Health and Disease 2011, 10:107http://www.lipidworld.com/content/10/1/107© 2011 Thornton et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the CreativeCommons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, andreproduction in any medium, provided the original work is properly cited.and stanols reduce plasma cholesterol levels, ostensiblyby inhibiting enterocytic cholesterol uptake throughcompetition with dietary and biliary cholesterol forabsorption [6-8]. The modification of hydrophobic plantstanols into a water-soluble phytostanol ester, where thephytostanols were covalently bonded to ascorbic acid(Vitamin C), was undertaken to combine the hypocho-lesterolemic properties of the phytostanol with thepotential benefits of an antioxidant [9,10]. The resultingchemical, disodium ascorbyl phytostanyl phosphate(DAPP) is a phytostanol analogue that exhibits a morepotent hypocholesterolemic effect than unesterified phy-tosterols and stanols alone [11-14].Data from studies examining the hypocholesterolemiceffects of DAPP also indicated that animals treated withthe compound experienced a dose-dependent decreasein body mass accumulation [11,15]. Further analysis ofthis compound-associated mass loss revealed that adi-pose tissue stores were reduced with no accompanyingreduction in lean body mass [16,17]. When previouslyobese animals were treated with 2% w/w dietary diso-dium ascorbyl phytostanyl phosphate in addition totheir high fat diet (45% kcal from fat), they immediatelybegan losing adipose tissue and within 8 days, theyachieved a statistically similar body mass to untreatedanimals on a low fat diet (10% kcal from fat). After 60days of compound administration, treated animalsexhibited a 41% decline in total body fat with no adverseeffects on organ mass, femur length, muscle mass orgross morphology; in addition, a significant increase inaerobic scope (VO2swim - resting metabolic rate) wasobserved. In effect, the compound turned previouslyobese mice on a high-fat diet into a low-fat diet pheno-type [17].Ingestion of unesterified plant sterols has beenreported to result in loss of adipose stores [18]. Researchalso indicates that mice on a high fat diet supplementedwith high doses of ascorbic acid accumulate significantlyless adipose tissue than their non-supplemented coun-terparts [19-22]. By examining the in vivo effects of dietsupplemented with ascorbic acid in the presence andabsence of phytosterols, we set out to establish whetherthe supplements have a synergistic effect in a diet-induced obesity mouse model, and to investigate possi-ble mechanisms for the supplement-induced mass loss.Materials and methodsAnimals and DietsAll animal studies were conducted with approval fromthe UBC Animal Care Committee. Male C57BL/6 mice(4 weeks old) were purchased from Charles Riverlaboratories (St. Constant, Quebec, Canada) and housedindividually with wood shaving bedding. The mice werekept at a constant temperature of 21°C ± 2°C in a 12 hrlight/dark cycle and had unrestricted access to food andwater throughout the period of the study. Animal mass,food mass and water intake were recorded once perweek. Metabolic assessment of all animals was initiatedon week 13; therefore only data from the first twelveweeks was used for mass accumulation analysis.Following an acclimatization period of eight days onregular mouse chow, C57Bl/6 mice were randomlyassigned into 4 groups (n = 8) and placed on the dietsfor 18 weeks. Using Research Diet’s 45 kcal% fat dietD12451 as a high fat control (HF), diet supplementswere milled into the HF control diet using either 1% w/w phytosterol/stanol mixture (85451; Sigma Aldrich,Oakville, ON; b-sitosterol ~76%, sitostanol ~13%, cam-pesterol ~8%, campestanol ~1%) for the PS diet, 1% w/w L-ascorbic acid (A5960; Sigma Aldrich, Oakville, ON)for the AA diet, or a combination of 1% phytosterol/sta-nols and 1% L-ascorbic acid for the PSAA diet.Oxygen ConsumptionAnimals were evaluated for whole animal metabolicconsumption using indirect flow-through calorimetryduring weeks 13-15. Measurement of oxygen consump-tion at rest (resting metabolic rate; RMR), and at maxi-mal swimming rate (VO2swim); were conducted on eachdietary group.Resting metabolic rate is defined as the lowest averageoxygen consumption at 21 ± 0.2°C over a 5 min periodduring the light phase (between 1000 and 1800 h) usingan open flow respirometry system. For RMR assessment,animals were placed in a sealed black Plexiglas 1075 mlchamber immersed in a 21 ± 0.2°C water bath as permethodology outlined in Thornton et al, 2007 [17].Briefly, outside atmospheric air was pushed through thechamber at a rate of 800 ml min-1 (0-1.5 l air pump,Rena Air 400A; Aalborg Mass Flow controller (0-5 l)). Asubsample of excurrent air was dried and scrubbed ofCO2 and passed through an oxygen analyser (BeckmanOM-11 polarographic oxygen analyzer) at a rate of 300ml min-1. Oxygen measurements were recorded eachsecond via a DI-710 A/D converter and the lowest 5min was corrected for pressure and temperature, andthen averaged to estimate RMR (Windaq DATAQ soft-ware). Immediately prior to all metabolic measurements,body mass was recorded to ± 0.1 g. Mice were notdenied food or water prior to respirometry measure-ments; however, as most food intake occurs nocturnally,it is an accepted practice to assume the animal isapproaching a post-prandial state near the end of aRMR assessment [23,24]. Each animal remained in thechamber for a minimum of 2 hours to ensure that RMRhad been achieved.To measure VO2swim, a glass funnel was suspendedover a water bath maintained at 20 ± 0.2°C. The animalsThornton et al. Lipids in Health and Disease 2011, 10:107http://www.lipidworld.com/content/10/1/107Page 2 of 14were introduced into the water bath and the funnel wassubmerged over the mouse to a predetermined height,leaving an air volume of 250 ml above the water level.Room air was introduced into the funnel at a rate of800 ml/min via a submerged air stone. The air stonewas positioned directly beneath the animal to encourageactive swimming. VO2swim was defined as the highestoxygen consumption averaged over 2 min of a 5 minswim trial. A subsample of excurrent air was evaluatedas described above.Body compositionWhole body fat measurements were carried out on a 7Tanimal MRI scanner (Bruker, Germany) on week 16 ofthe study. Unanesthetised mice were placed inside aPlexiglas restrainer, and the restrainer was positionedinside the bore of the magnet. NMR signal from theentire body was acquired with a quadrature volume RFcoil tuned to 300MHz. A standard CPMG sequence (TE= 2.377 ms, TR = 10 s) was used to acquire 256 echoesfrom which the T2 decay curve was extracted. Thedecay curves were then fit to a double exponential func-tion using software procedure developed in house withIgor (WaveMetrics, OR). The ratio of lean tissue/bodyfat expressed as weight/weight is then calculated fromthe NMR data as described previously [25].Food transit timeFood transit time was measured by timing the fecalappearance of Sudan Red III dye administered in thediet as per Toloza et al, 1991 [26]. Briefly, 300 mgSudan red III dye was dissolved in 250 mL of acetone,and 300 mg of each diet was placed in a glass beakerand covered with the Sudan red/acetone mixture. Thedyed rations were placed in a fume hood for 72 hoursof evaporation to eliminate the acetone.To assess the effect of chronic exposure to a HF dietwith or without supplements on food transit time, onweek 18 of the experiment, all mice were switched tothe non-additive HF control diet for 24 hours. At 5 am,animals were offered the HF control Sudan Red III dyeddiets for two hours, followed by a collection of feces fortwenty hours during ad libidum feeding of HF controlration. Fecal samples from each collection time pointwere dried for a minimum of 48 hours at 60°C andstored at -20°C until analysis. Fecal pellets were groundusing a mortar and pestle and a ~100 mg aliquot wasplaced in a screw top test tube, vortexed with acetone ina 50:1 w/v ratio and allowed to stand at RT for 30 min.The samples were centrifuged at 3000 rpm for 3 minand the quantity of dye in the acetone was assessedspectrophotometrically at 504 nm. The total amount ofdye eliminated by each mouse during the 20 h collectionperiod was taken to be 100%, and the data from eachtime point are expressed as a percent excretion of thetotal dye released over the twenty-hour collection per-iod. Curves were then fit to a sigmoidal logistic functionusing Systat and individual times for 10% through 100%dye excretion were calculated. Values were indexed todiet and analysed using a one-way ANOVA; significantlydifferent group means were identified using Tukey Test.To establish the acute effect of dietary components onfood transit time, age-matched C57Bl/6 mice raised onnormal chow were randomly assigned a dietary groupand were exposed to the experimental diet for 72 hoursprior to food transit time analysis using abovemethodology.Digestive efficiencyDigestive efficiency is a measure of the amount of che-mical energy absorbed from the diet. As the diets haveslight differences in caloric value and sterol content, thevalues of fecal energy outputs were corrected for con-sumed phytosterol/stanol amounts. Diets were relativelyisocaloric between the HF control (4.73 kcal/g), AA(4.69 kcal/g), PS (4.68 kcal/g) and PSAA (4.65 kcal/g)rations. As it is assumed that phytosterols are notabsorbed, the caloric content of PS is not included inthe above caloric content. Feces were collected over aperiod of 72 hr by replacing the shavings in each cagewith a metal grid to facilitate fecal collection. Collectedfecal material was dried overnight in an oven at 60°C,then stored at -20°C until analysis. Fecal energy contentwas assessed using a bomb calorimeter (Adiabaticcalorimeter 1241, Parr Instrument) and corrected forphytosterol content. Digestive efficiency was calculatedfor each animal as the difference between gross energyconsumed and fecal energy output over a 72-hourperiod.Statistical analysisStatistical analysis was conducted using Sigmastat(Systat for Windows; v. 5.02). To test effects of dietand supplement, we used a one-way ANOVA; signifi-cantly different group means were then separatedusing a Tukey Test or Dunnett’s Method as appropri-ate. Using Sigmaplot, passage rate analysis was con-ducted by curve fitting the data from each individualanimal and calculating individual output values for 10-100% dye excretion. Values were indexed to diet andanalysed using a one-way ANOVA in Sigmastat; signif-icantly different group means were identified using anANOVA and a post-hoc Tukey Test; observed differ-ences between supplement groups and high fat controlwere reported. Values with p < 0.05 were consideredto be significant.Thornton et al. Lipids in Health and Disease 2011, 10:107http://www.lipidworld.com/content/10/1/107Page 3 of 14ResultsGrowth curvesOver the first 12 weeks of the study, animals fed a HFdiet supplemented with a combination of AA and PSexhibited a decrease in mass accumulation when com-pared to control (PSAA group, n = 8, ANOVA followedby a post-hoc Tukey; p < 0.05, Figure 1a). Animals inthe PSAA group exhibited a lower mass by week 2 andthis difference became significant by week 7 of the study(n = 7; p < 0.05). There was no significant difference incaloric intake or water intake between the dietarygroups throughout the growth assessment period (12weeks; ANOVA, data not shown).Oxygen ConsumptionMetabolic assessment was conducted on animals duringweeks 13-15 of chronic exposure to a HF diet with orwithout supplements. Dietary supplementation with PS,05101520250 20 40 60 80Time (days)Mass Accumulation (g) HF*HF+ PSAA*HF 2007  Figure 1 Mass accumulation of male C57bBl/6 mice exposed to a high fat diet with or without supplements. 1a) Animals in thephytosterol+ascorbic acid (PSAA) group exhibited a reduction in mass accumulation by week 2 and the difference became statisticallysignificant by week 7 of the study (n = 7; ANOVA p < 0.05). 1b) Mass accumulation over time for C57Bl/6 animals consuming a high fat dietwith ascorbic acid and phytosterol supplementation (this study) in comparison to animals consuming a high fat control diet with or withoutdisodium ascorbyl phytostanyl phosphate(17). Mice chronically exposed to a high fat diet supplemented with 2% w/w phytostanol esterified withascorbic acid show a greater decrease in mass accumulation than those consuming a high fat diet supplemented with unesterified phytosterolsand ascorbic acid (1% w/w of each component). * indicates data obtained from this study.Thornton et al. Lipids in Health and Disease 2011, 10:107http://www.lipidworld.com/content/10/1/107Page 4 of 14AA or PSAA did not result in significant differences ofRMR or VO2max when compared with HF control (n =8; ANOVA).Body CompositionThe ratio of lean tissue/body fat expressed as weight/weight was measured for each animal using MRI. Smallbut significant differences were observed between the %body fat and lean-to-fat ratios in animals in the PS andPSAA groups when compared to HF control (n = 8;ANOVA, post-hoc Tukey; % body fat values HF 47.37%;AA 46.66%; PS 45.32%; PSAA 45.70%; Figure 2).Fecal Caloric ContentFecal caloric content was compared both within theacute and chronic exposure groups as well as betweeneach dietary group. After corrections were applied toaccount for fecal phytosterol content, we did notobserve a significant difference between the caloric con-tent of the feces between the diets in either the chronicor acute exposure groups (n = 8; ANOVA). When fecalcaloric content was compared within each dietary group,we observed a significant reduction in fecal caloric con-tent with chronic exposure to the HF (p = 0.0002), AA(p = 0.003) and PS (0.004) diets (n = 8; t-test). ThePSAA group did not demonstrate a significant reductionin fecal caloric content after chronic exposure to thesupplement (chronic 15.34 ± 0.47 vs acute 16.13 ± 1.06;p = 0.09; Figure 3).Food Transit TimeA comparison of the HF control group to the groupscontaining additives was undertaken for both the acuteand chronic diet protocols in order to assess the effectof supplements on food transit time. Each dietary groupwas also statistically assessed to establish the effect ofchronic exposure to the diet on food transit time. Spec-trophotometric assessment of fecal dye content wasexpressed as % excretion over the 20-hour assessmentperiod. Based on the assumption that dye consumptionwas complete at the midpoint of the 2-h pulse, dyerecovery times for 10% through 100% excretion werecalculated for each animal by curve fitting using a sig-moidal logistic function. Age-matched C57Bl/6 mice did**Figure 2 Body fat significantly decreases with the addition of 1% phytosterol to a high fat diet when compared to HF control (HF n =7, all other groups n = 8; ANOVA followed by a post-hoc Dunnett’s Method). Animals were assessed using magnetic resonance imagingafter 16 weeks on the dietary supplements.Thornton et al. Lipids in Health and Disease 2011, 10:107http://www.lipidworld.com/content/10/1/107Page 5 of 14not exhibit any differences in food transit time after anacute 72-hour exposure to a HF diet with supplementswhen compared to animals exposed to the HF controldiet (n = 7, ANOVA; Figure 4a).After 18 weeks of exposure to PS, AA or PSAA, diet-ary supplements were withdrawn for 72 hours in orderto assess the chronic effect of additives on transit time.Placing all animals on the HF diet alone eliminated apossible intrinsic effect of the dietary supplements onpassage rate. Animals in PS and PSAA exhibited signifi-cant differences in transit time when compared to theHF control (ANOVA with post hoc Tukey Test; Figure4b). The 50% dye recovery values (mean ± SD) for eachdiet were eliminated at 9.00 ± 2.17 h (HF; n = 7); 8.39 ±2.22 h (AA; n = 7); 5.2 ± 1.12 h (PS; n = 3), and 7.02 ±2.47 (PSAA; n = 4). Some animals in the PS and PSAAgroups did not consume any of the Sudan Red III dyedration during the 2-h pulse, therefore the n number islower in these groups.When the acute food transit time was compared tochronic food transit time for each dietary group, no sig-nificant change in passage rate was observed for animalson the HF or AA diets (n = 7; ANOVA; Figure 5a).However, a significant decrease in food transit time wasobserved for animals in both the PS and PSAA supple-mented diet groups (n = 3 and 4 respectively; ANOVAfollowed by a post-hoc Tukey Test, Figure 5b).Food consumption and fecal outputWeekly average food consumption and gross energyintake did not differ significantly between dietary groups(n = 8; ANOVA). Experimental manipulations (meta-bolic assessment, swim challenge, fecal collection grids)were associated with a temporary reduction of food* **Figure 3 Fecal caloric content in age-matched animals exposed to the diet for 72 hours (acute - closed bars) or 17 weeks (chronic -open bars). A significant decline in caloric content of the feces was observed in animals chronically exposed to the diet in the HF, AA and PSgroups when compared to an acute 72-hour exposure in age-matched individuals (n = 7; t-test within each dietary group). Asterisks indicate asignificant decline in fecal caloric content is observed in animals exposed to a diet for 17 weeks when compared to acute dietary exposure.After caloric content was corrected for estimated phytosterol content, no significant difference was observed between the diets within eachexposure.Thornton et al. Lipids in Health and Disease 2011, 10:107http://www.lipidworld.com/content/10/1/107Page 6 of 14   Figure 4 Food transit time of age-matched C57Bl/6 mice after an acute (72-hour) and chronic (18 week) exposure to a high fat dietwith or without ascorbic acid or phytosterol supplements. 4a) Acute exposure to dietary supplements did not alter food transit time whencompared to the high fat control (n = 7; ANOVA). 4b) Animals chronically exposed to PS and PSAA supplements exhibited significant differencesin transit time of a HF meal when compared to the HF control (ANOVA with post hoc Tukey Test; n = 7 for HF and AA; n = 3 for PS, n = 4 forPSAA).Thornton et al. Lipids in Health and Disease 2011, 10:107http://www.lipidworld.com/content/10/1/107Page 7 of 14intake; therefore, mass accumulation and food consump-tion data are reported only for the first 12 weeks of thestudy.Fecal output was assessed under three different condi-tions. Acute exposure to HF diet with or without sup-plements in age-matched C57Bl/6 mice indicated thatall diets containing supplements resulted in a significantincrease in fecal output when compared to the HF con-trol (n = 7; ANOVA followed by a post-hoc Dunnett’sMethod; Figure 6). In animals fed a 1% w/w phytosteroldietary supplement, a correction factor accounting forthe undigested phytosterol component is applied. Basedon the mean daily food consumption and fecal output(~3 g and 300 mg, respectively) the fecal mass in ani-mals consuming a diet containing phytosterol isexpected to be to be ~10% higher due to the non-diges-tible component.Animals exposed to the experimental diets with sup-plements for 17 weeks showed no significant differencesin fecal output when compared to the HF control (n =7, ANOVA; Figure 7). When the dietary supplementswere removed for 72 hours and animals were fed a HFdiet, a significant decrease in fecal output (>40%) wasobserved in the PS and PSAA groups (ANOVA followedby a post-hoc Dunnett’s Method, Figure 8).DiscussionMass accumulation and fecal energy lossOur data indicate that in a diet-induced obesity C57Bl/6mouse model, chronic administration of a high fat dietwith 1% w/w phytosterol and 1% w/w ascorbic acid(PSAA) results in decreased mass accumulation whencompared to either the HF control diet, diets supple-mented with 1% w/w ascorbic acid (AA), or diets sup-plemented with 1% phytosterol (PS) alone (Figure 1a;the decrease in PSAA mass accumulation was significantfrom weeks 7-12). The reduced rate of mass gain is notaccompanied by decreased food or water intake, differ-ences in resting metabolic rate, or alteration of maximaloxygen consumption (VO2max) when compared to HFcontrol animals (data not shown; n = 8 for each dietarygroup; ANOVA). Interestingly, the decrease in massaccumulation is not as profound as that observed in ourprevious studies where the high fat diet was supplemen-ted with 2% w/w phytostanol esterified with ascorbicacid (DAPP; Figure 1b), suggesting that esterification ofthe components potentiates the mass loss mechanism[17]. The mean difference in absolute mass betweenPSAA and HF control for the six-week period is 10.05%,and the mean difference in mass accumulation is 21.6%(i.e. the PSAA group gained on average 21% less masseach week from weeks 7-12 than the HF control group).01020304050607080901000 2 4 6 8 10 12 14 16 18Time (hours)% Sudan Red Excreted in FecesHFHF AAHF PSHF PSAA01020304050607080901000 2 4 6 8 10 12 14 16 18Time (hours)% Sudan Red Excreted in fecesHFHF AAHF PSHF PS AAFigure 5 Food transit time of a HF diet in animals chronicallyexposed to a high fat diet with or without ascorbic acidsupplements is compared to acute (72 hr) exposure transittimes. 5a) In the HF and AA groups, chronic exposure to diets didnot alter food transit time when compared to acute exposure tothe diet for 72 hours in age-matched C57Bl/6 mice (n = 7; ANOVAfollowed by a post-hoc Tukey Test). 5b) Chronic exposure to a HFdiet supplemented with phystosterol alone or phytosterol withascorbic acid significantly reduced food transit time whencompared to acute exposure to the diet for 72 hours in age-matched animals (n = 7; significant differences between PS chronicand acute were observed at 20 - 90%; PSAA chronic and acute at80 and 90%; ANOVA followed by a post hoc Tukey Test).Thornton et al. Lipids in Health and Disease 2011, 10:107http://www.lipidworld.com/content/10/1/107Page 8 of 14If we look at our 2007 DAPP study data for the compar-able time period, the mean difference in absolute massbetween the 2% DAPP and HF control was 41%, whilethe mean difference in mass accumulation between thetwo groups for weeks 7-12 was 67.9%. In animals on the2% DAPP-supplemented diet, this reduction in massaccumulation was correlated with adipose tissue reduc-tion. Magnetic resonance spectroscopy and imaging stu-dies of the mice at week 16 of the current studyrevealed that animals in the PS and PSAA groupsexperienced a slight but significant decline in adiposetissue mass when compared to the HF control (Figure2), but only the PSAA group exhibited an accompanyingreduction in mass accumulation. Our data are in con-currence with Ebine et al (2006), where supplementationwith 2% w/w DAPP resulted in a significant reduction inmass accumulation in hamsters, whereas supplementa-tion with 1% w/w unesterified phytostanol alone did notalter mass accumulation [16].Ebine and colleagues also indicated that fecal energyoutput (adjusted for the phytostanol component) wasnot significantly different in animals administered a phy-tostanol-supplemented diet when compared to the con-trol diet [16]. Our findings indicate that within both theacute (72 hour) and chronic (17 weeks) exposure experi-ments, supplementation with PS, AA or PSAA did notresult in a significant alteration of fecal caloric contentwithin each exposure experiment when compared to theHF control (Figure 3). However, a comparison of fecalenergy output from acute exposure vs. chronic exposurewithin the same dietary groups revealed a significantdecrease in fecal energy after 17 weeks of exposure tothe HF control, PS and AA diets. A non-significantdecrease in fecal caloric content was also noted in thePSAA group (t-test; p = 0.09). These data indicate thatlong term exposure to a high fat diet, regardless of sup-plement, results in a decrease in fecal energy and sug-gests that the animals are absorbing a greater portion of020406080100120140160180200HF AA PS PSAAFecal output - acute (% of control) PhytosterolFecal output***Figure 6 Fecal output of age-matched mice after a 72-hour acute exposure to HF diets with or without supplement. As phytosterols areassumed to be 100% excreted, fecal output for animals in the PS and PSAA groups were corrected to account for the 1% of dietary intake ofnon-digestible phytosterol (PS accounts for ~ 10% of fecal output, based on a daily consumption of 1% of 3 g chow and average daily fecaloutput of 300 mg). Acute exposure to all HF diets containing supplements resulted in a significant increase in fecal output when compared toHF control (n = 7; ANOVA followed by a post-hoc Dunnett’s Method).Thornton et al. Lipids in Health and Disease 2011, 10:107http://www.lipidworld.com/content/10/1/107Page 9 of 14the dietary caloric content with chronic HF exposure[27]. Chronic exposure to a high fat diet has beenshown to enhance intestinal cell proliferation, height ofintestinal villi and to increase enterocytic migration ratefrom crypt to villus [28-30]. The reduced rate of massaccumulation in the PSAA group may be partly due toamelioration of the increased absorptive capacity that isassociated with chronic dietary HF exposure.Food transit time and fecal outputLong-term exposure to dietary PS supplementationresulted in significant changes in energy assimilationand digestive efficiency. In the 72-hour exposure experi-ment, acute exposure to dietary supplements did notalter food transit time (Figure 4a), but did result in anincreased fecal output in all groups when compared tothe HF control (Figure 6). Taken together, these datasuggest that short-term exposure to the dietary supple-ments results in some degree of interference with diet-ary uptake, as evidenced by a greater fecal mass with noalteration in passage rate (data are summarized in Table1). Acute dietary PS, AA or PSAA supplementation didnot alter the caloric content of the feces when comparedto the HF control (Figure 3, data corrected for estimatedfecal phytosterol output; n = 8, ANOVA), therefore thenet effect of acute exposure on the absorptive processappears to be an overall decrease in energy assimilationof a HF diet when it is accompanied by PS, AA orFigure 7 Fecal output after 17 weeks of chronic exposure to a high fat diet with or without phytosterol or ascorbic acid. Asphytosterols are assumed to be 100% excreted, fecal output for animals in the PS and PSAA groups were corrected to account for the 1% ofdietary intake of non-digestible phytosterol (PS accounts for ~ 10% of fecal output, based on a daily consumption of 1% of ~3 g chow andaverage daily fecal output of ~300 mg). Dietary supplements did not result in a significant difference in fecal output when compared to thehigh fat control diet (n = 7; ANOVA followed by a post-hoc Dunnett’s Method).Thornton et al. Lipids in Health and Disease 2011, 10:107http://www.lipidworld.com/content/10/1/107Page 10 of 14PSAA. In animals chronically exposed to the HF dietwith supplements, neither the fecal output (Figure 6),nor the caloric content of the feces (Figure 3) differedfrom the HF control, suggesting that the animals haveaccommodated the effects of the supplements over thecourse of the chronic exposure (n = 7; ANOVA).We hypothesize that in the chronic exposure animals,increased fecal output and thus decreased energy uptakestimulates intestinal remodeling to compensate for theenergy. When supplements were removed from the dietand all animals were placed on the HF control diet for72 hours, no significant difference in the food transittime of HF control or AA supplemented diet groupswas observed, indicating that chronic exposure to 1%AA supplementation did not induce changes in theintestine that would result in altered passage rate (Fig-ure 7a). Interestingly, animals chronically exposed todiets containing PS or PSAA exhibited a significant andprofound decrease in the transit time of a HF diet (Fig-ure 7b). In the same animals, fecal output dropped bymore than 40% (Figure 8). In an animal chronicallyexposed to PS supplements, these data describe a**Figure 8 Fecal output from animals switched to a high fat diet for 72 hours after 17 weeks of chronic exposure to a high fat diet withor without phytosterol or ascorbic acid. As phytosterols are assumed to be 100% excreted, fecal output for animals in the PS and PSAAgroups were corrected to account for the 1% of dietary intake of non-digestible phytosterol (PS accounts for ~ 10% of fecal output, based on adaily consumption of 1% of 3g chow and average daily fecal output of 300 mg). Chronic exposure to phytosterol supplements in the dietresulted in a significant decrease in fecal output when additives were withdrawn (n = 7; ANOVA followed by a post-hoc Dunnett’s Method).Thornton et al. Lipids in Health and Disease 2011, 10:107http://www.lipidworld.com/content/10/1/107Page 11 of 14situation whereby the HF diet is in contact with theintestinal absorptive surface for significantly less time,yet a significantly greater proportion of the mass of themeal is absorbed when compared to acute exposure, asevidenced by a reduction in fecal output. These findingssuggest that chronic exposure to PS in the diet altersthe intestine and results in upregulation of its absorptivecapacity. The current opinion in the literature on themechanism of phytosterol’s hypocholesterolemic proper-ties suggests that physical interference of cholesterolabsorption through either competitive positioning in themicelle, disturbance of micellar formation, or a combi-nation of both plays a major role [10,31-33]. This modeof action may also be responsible for alteration of lipiduptake and result in a proliferation of intestinal trans-porters to counteract the effect of dietary phytosterol onenergy procurement. When PS supplementation isremoved from the diet, a significant and profoundreduction in fecal output and a decrease in transit timemay be partly explained by a potential increase in sur-face area and elevation of uptake mechanisms in theintestinal tract. Further studies involving histologicaland immunohistochemical analysis of the intestinal tractwould be necessary to test this hypothesis.There are a number of studies reporting that animalson a high fat diet supplemented with high doses ofascorbic acid accumulate significantly less adipose tissuethan their non-supplemented counterparts [20,21]. Thisfinding is not unique to ascorbic acid, but is alsoobserved in response to other antioxidants. In an obesehamster model, co-administration of a high fat diet withExtramel microgranules (a melon juice extract coatedwith palm oil and rich in antioxidants and particularlysuperoxide dismutase) prevented obesity in the high fat-fed hamsters by decreasing body weight, abdominal fat,triglyceridemia, insulinemia, insulin resistance, liverlipids, and nonalcoholic steatohepatitis and preventingadipokine imbalance [34]. It is also thought that theantioxidant properties of the Mediterranean diet contri-bute significantly to its associated decreased risk of obe-sity and reduced levels of metabolic syndrome whencompared to an isocaloric Western diet [35]. Severalstudies have reported a significant inverse relationshipbetween plasma vitamin C concentrations and degree ofobesity, further supporting a correlation between adi-pose tissue accumulation and ascorbic acid supplemen-tation [22,36,37]. Our data did not support the findingsof Campion et al, as the 1% AA supplementation didnot result in a decrease in mass accumulation in DIOmice [20,38]. However, the potentiation of AA absorp-tion and thus higher circulating plasma levels may havebeen affected by the presence of PS in the diet and itspossible effect on the absorptive capacity of the intes-tine, which could lead to the observed increased efficacyof the PSAA combination. Esterification of ascorbic acidat position 2 protects vitamin C from destruction byoxidation and may lead to even higher circulatingplasma levels, which may assist in elucidating themechanism associated with the esterified ascorbic acid/phytostanol compound [39,40].In our previous study using the dietary supplementdisodium ascorbyl phytostanyl phosphate (DAPP), weobserved that animals on a high fat diet with supple-ment exhibited a significant dose-dependent reductionin mass accumulation over time when compared to theHF control group [17]. In the current study, the unes-terified components of DAPP (ascorbic acid and a phy-tosterol/phytostanol mixture) were administeredseparately and in combination in the presence of a highfat diet. Although the combination of supplementsresulted in a significant decrease in mass loss whencompared to the high fat diet alone or single-additivesupplementation, the decrease was not as profound asthat observed in the DAPP study. The current study didTable 1 Summary of findings outlining significant differences in food transit time and fecal output for mice exposedto a HF diet with or without phytosterol or ascorbic acid supplement for 72 hours (ACUTE) or a 17-20 week exposure(CHRONIC)HF AA PS PSAAFOODTRANSIT TIMEFECALOUTPUTFOODTRANSIT TIMEFECALOUTPUTFOODTRANSIT TIMEFECALOUTPUTFOODTRANSIT TIMEFECALOUTPUTACUTE to HF control n/a n/a NSD ↑ NSD ↑ NSD ↑CHRONIC to HF control Did not test n/a Did not test NSD Did not test NSD Did not test NSDCHRONIC on HF to HFcontroln/a n/a NSD NSD ↓ ↓ ↓ ↓CHRONIC on HF toACUTE (paired)NSD NSD NSD NSD ↓ ↓ ↓ ↓Animals in the chronic exposure group were switched to a HF control diet for 72 hours; these animals are referred to as “CHRONIC on HF”. Dietary groups withsupplements were compared to the HF control within each exposure experiment and assessed for statistically significant differences. To evaluate long-termchanges to digestive efficiency, all dietary groups in the “CHRONIC on HF” exposure were compared values obtained from animals acutely exposed to the samedietary groups ("ACUTE”). HF control comparisons to HF control are deemed not applicable (n/a); arrows indicate values are statistically lower or higher than HFcontrol or dietary pair group. NSD = non-significant difference.Thornton et al. Lipids in Health and Disease 2011, 10:107http://www.lipidworld.com/content/10/1/107Page 12 of 14not use the same species of phytostanols that wereemployed in the manufacture of DAPP; therefore wecannot make a direct comparison of esterified phytosta-nyl vs unesterified phytostanol in the presence of ascor-bic acid. In addition, the phosphodiester bond mayafford the compound greater protection during the acid-labile digestive process and allow for absorption in amore efficacious form. However, the data support thehypothesis of decreased mass loss associated with diet-ary supplementation with plant sterols and antioxidants,and provide some insight into possible mechanisms ofthese compounds on energy assimilation in the develop-ment of obesity in high fat fed mice.AcknowledgementsThe authors would like to thank Julian Kaye and the staff at the AnimalResource Unit for their technical assistance. The expertise, patience and skillof Andrew Yung and Jenny Tso at the University of British Columbia’s MRIResearch Centre are gratefully acknowledged. The funding for this researchwas provided by a Canadian Institutes of Health Research grant to KMW andSJT.Authors’ contributionsSJT designed the experiments, carried out the studies, completed the dataanalysis and wrote the manuscript. ITYW carried out the studies andcompleted the data analysis. RN carried out the studies and completed thedata analysis. PK carried out the studies and completed the data analysis.KWM designed the experiments, completed the data analysis and revisedthe manuscript. All authors have read and approved the final manuscript.Competing interestsThe authors declare that they have no competing interests.Received: 28 April 2011 Accepted: 28 June 2011Published: 28 June 2011References1. Hevener AL, Febbraio MA, the Stock Conference Working Group: The 2009Stock Conference Report: Inflammation, Obesity and Metabolic Disease.Obes Rev 2010, 11:635-44.2. Toyoda T, Kamei Y, Kato H, Sugita S, Takeya M, Suganami T, Ogawa Y:Effect of peroxisome proliferator-activated receptor-alpha ligands in theinteraction between adipocytes and macrophages in obese adiposetissue. Obesity (Silver Spring) 2008, 16:1199-1207.3. Holvoet P: Relations between metabolic syndrome, oxidative stress andinflammation and cardiovascular disease. Verh K Acad Geneeskd Belg 2008,70:193-219.4. Ross R: Atherosclerosis–an inflammatory disease. 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Hidiroglou M, Batra TR, Zhao X: Comparison of vitamin C bioavailabilityafter multiple or single oral dosing of different formulations in sheep.Reprod Nutr Dev 1997, 37:443-448.doi:10.1186/1476-511X-10-107Cite this article as: Thornton et al.: Dietary supplementation withphytosterol and ascorbic acid reduces body mass accumulation andalters food transit time in a diet-induced obesity mouse model. Lipids inHealth and Disease 2011 10:107.Submit your next manuscript to BioMed Centraland take full advantage of: • Convenient online submission• Thorough peer review• No space constraints or color figure charges• Immediate publication on acceptance• Inclusion in PubMed, CAS, Scopus and Google Scholar• Research which is freely available for redistributionSubmit your manuscript at www.biomedcentral.com/submitThornton et al. Lipids in Health and Disease 2011, 10:107http://www.lipidworld.com/content/10/1/107Page 14 of 14


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